Regulation of Flavonoid Biosynthesis (in seeds and other ... · Flavonoids in seeds Flavonols: ... Field tests for pollination Hoballah et al., 2007 Diurnal butterflies Diurnal hymenopteran

Regulation of Flavonoid Biosynthesis

(in seeds and other tissues)

Koes et al., 2005

Flavonoids in seeds

- Found in most seeds and grains- Major ones are:

a. Flavonolsb. Anthocyaninsc. Phlobaphenesd. Isoflavonese. Proanthocyanidins (PAs, also condensed tannins)

Flavonoids in seedsFlavonols:- contribute to seed pigmentation mainly as co-pigments with anthocyanins- most frequent are glycosides derivatives (glucose and rhamnose at C3 and C7)

Anthocyanins:- in seeds of both mono and dicots- barley grains, bean seed coat, aleurone layer in maize

Flavonoids in seeds

Phlobaphenes (deoxyflavonoids):- red polymers of flava-4-ol precursors found in maize and other monocots in the seed coat

Isoflavones:- colorless compounds, occur at the subfamily Leguminosae (soybean)- major metabolites in the embryo and seed coat

Flavonoids in seeds

Proanthocyanidins (PAs, also condensed tannins):

Described in detail (in Arabidopsis)

Flavonols and PAs in Arabidopsis seeds


- Arabidopsis seeds accumulate only flavonols and PAs (50/50)

- Flavonols present in testa (seed coat) and the embryo

- Quercetin (quercetin-3-O-rhamnoside) is the major flavonol

Lepiniec et al., 2005


PAs in Arabidopsis seeds

- PAs accumulate in the seed coat

- Protect the embryo and endosperm

- Oxidation of PAs during seed desiccation-brown pigment to mature seeds


- The seed coat results from differentiation of the integument and chalazal tissue after fertilization

- The seeds have an outer and inner integument

- PAs are synthesized in the endothelium, the inner most cell layer of the inner integument


Endothelium(PAs synthesis)

Altered seed pigmentation in Arabidopsis:the transparent testa (tt) mutants

Altered seed pigmentation in Arabidopsis:the transparent testa (tt) mutants

PA metabolism in Arabidopsis seeds:tt10- PAs oxidative polymerization(a polyphenol oxidase)

- PAs in Arabidopsis testa are the flavan-3-ol 2,3-cis-(-)-epicatechin oligomers

- PAs are colorless polymers localized to the vacuole

- During seed desiccation, vacuole burst will localize a polyphenol oxidase enzyme (TT10?) with PAs in the appoplast and this will result in browning

PA metabolism in Arabidopsis seeds:tt10- PAs oxidation

Gene expression localizes with PAs and flavonols synthesis

TT10 can oxidize epi-catechin and quercetin

Flavonoids in Arabidopsis seeds- Metabolon formation?

Winkel BS. 2004

Regulation of PAs biosynthesis in Arabidopsis seeds


- TT1 (zinc-finger) and TT16 (MADS box), may control cellular differentiation leading to PA accumulation and not PA biosynthesis directly

- Needed for PA accumulation in the endothelium (region 2) but not in micropyle and chalaza area


- TT2- R2-R3-MYB protein- TT8- bHLH protein- TTG1- WDR protein- TTG2- WRKY transcription factor

Plant MYB FactorsStracke et al., 2001

Plant Basic Helix-Lop-Helix Factors

- More than 160 members in Arabidopsis

- Contain a bHLH domain that is involved in binding DNA

Regulation of PAs biosynthesis in Arabidopsis seedsTT2, TT8 and TTG1 regulate BAN but also other flavonoid pathway genes

Regulation of PAs biosynthesis in Arabidopsis seedsTT2, TT8 and TTG1 regulate BAN but also other flavonoid pathway genes

endothelium

Chalaza (pigment strand)

micropyle

Regulation of PAs biosynthesis in Arabidopsis seedsTT2, TT8 and TTG1 forming the MBW complex

- TT2- R2-R3-MYB protein

- TT8- bHLH protein- TTG1- WDR protein


- R2-R3 MYBS can bind directly to DNA, either on their own or in the presence of bHLH proteins- bHLH binding alone was never demonstrated- Interaction MYB-bHLH was detected a few times (N-termini of bHLH interacts with R3 repeat of MYBS)- WDR interact with MYB and bHLH


- a ternary complex is able to bind the BAN promoter

Regulation of PAs biosynthesis in Arabidopsis seedsWhat regulates TT8?

+ TTG1

- TTG1, TT2 and PAP1 regulate TT8- TT8 activates itself to synchronize its levels with those of its partners in the MBW complex

Baudry et al., 2006

Epidermal cell fate, seed-coat development and flavonoid biosynthesis are linked in Arabidopsis

- The above relationship first became apparent with the discovery of the transparent testa glabra1(ttg1) mutants

- The ttg mutants typically:- produce few or no trichomes- have an aberrant pattern of root hair initiation- produce yellow seeds (reduction in PAs)


wt wtttg1 ttg1

Broun, 2005


- The ttg mutants also:

- Mucilage production in the seed coat is impaired- Anthocyanins in leaves, stems do not accumulate or are reduced in levels


Single repeat MYBs (R3), lack an activation domain


Regulatory genes controlling pigment synthesis in Arabidopsis, petunia and maize

What pathway and in what tissue will be largely determined by expression patterns of the regulators, their interactions and the cis-elements on the promoters of structural genes

Activity of a similar complex in Morning Glory

Morita et al., 2006

Regulation of flavonol metabolism and not anthocyanins by MYB sub-group 7

Sub-group 7

Stracke et al., 2005, 2007




- In the flavonoid pathway a single target gene is controlled by multiple transcription factors- The factors belong to the same transcription factor family and share the same DNA binding domain- Different expression patterns (spatial, temporal and stimulus responsive transcript accumulation patterns) could explain the regulation of production of a certain phenylpropanoid

Regulation of floral pigmentation intensity & patterning

- Many color patterns provide guides that direct pollinators towards the reproductive organs and the source of nectar within flowers

- In snapdragon - yellow is an Aurone and magenta is Anthocyanin

Schwinn et al., 2006


- Some natural isolates of snapdragon have different patterning, with increased pigmentation in regions of the epidermis overlaying the vascular strands of the petal

- Mutation in regulatory genes do not abolish pigmentation (as in structural genes), but change the pattern of pigmentation in the flowers

Mutations in snapdragon regulatory genes

Delila - pigmentation in the corolla lobes (bHLH)

Mutabilis - pigmentation in the corolla lobes

Rosea - pattern and intensity of pigmentation in both lobes and tubes

Venosa (dominant) - pigmentation of the epidermis overlaying the veins in both lobes and tubes

rosea and venosa mutants

- 2 mutant alleles for rosea (colorata and dorsea)- weak anthocyanin production

- Venosa: could be seen on the ros backgrounds only (Ros+ epistatic to Ve+)- pigment over the veins


The Rosea Locus (Ros1 & Ros2 are linked genes)

Three different MYB genes isolated

Loss of function in Ros1

Reduced expression of Ros1 and loss of functionRos2

The Rosea genes activate anthocyanin biosynthesis in a rosdor background


The Venosa gene

Ros petal Schwinn et al., 2006

Ros1, Ros2 and Ve have similar but not identical targets


- Ros1 increases LBGs expression (F3H, DFR, ANS & UFGT)

- Ros2 regulates the expression of F3'H

- Ve presence increase CHI, F3H, F3'H, FLS, ANS, UFGT and AT

Interaction of Ros1, Ros2 and Ve (MYB) with Delila (Del) and Mutabilis (Mut) bHLH factors

A: roscol (only Ros2 expressed, Ros1 missing)

B: roscol del (Ros2 interacts with Del but not with Mut that needs to be activated by the missing Ros1)

C: roscol Ve+

D: roscol Ve + del (Ros2 interacts with Del in the lobes and Ve can interact with Mut)


Variation in anthocyanin patterns in flowers of different snapdragon species results principally from variation in activity of MYB- related genes



- A small 3 members family in snapdragon controls the pattern and intensity of floral pigmentation

- The 3 genes are not functionally equivalent (target genes and expression pattern different)

- Seem to arise by gene duplication and sub-functialization


- Variation in anthocyanin production is attributed to MYB genes in flowers of petunia and snapdragon, berry skin color in grape and potato tuber

- The above suggests that the same route was followed independently on a number of occasions

- Altering regulatory proteins rather than structural genes is more rapid

ANTHOCYANIN2 (AN2), a MYB factor detrmaining flower color variation and pollinator attraction

- Specific sets of floral traits are often associated with a particular group of pollinators (adaptation)

- Adaptation:- a single pollinator- some orchids- one class of pollinators (long-tongued nocturnal hawk moth- multiple pollinator types

Adaptation of flower by:flower morphology, scent, nectar production, color

Hoballah et al., 2007

ANTHOCYANIN2 (AN2), a MYB factor determining flower color variation and pollinator attraction


- The genus petunia is pollinated with either hawk moth or bees

- Petunia axillaris- white flowers with long and narrow corolla tubes, emits a large amount of volatiles at night and contains a large amount of nectar.Pollinated by hawk moth

- Petunia integrifolia- violet-reddish flower color, short corolla tube containing low amounts of nectar and emitting small amounts of volatiles- Pollinated by bees


Petunia axillaris

Petunia integrifolia



- P. Axillaris carries mutations in the AN2 gene- Loss of color arose multiple times-



Field tests for pollination


Diurnal butterflies

Diurnal hymenopteran(bees, wasps, ants)

Hawk moth could not be assessed

Green house tests for pollination

Wild species

Introgression lines

Green house tests for pollination in transgenic plants overexpressing AN2 in the white axilllaris petunia

- Single gene-mediated shift in pollinator attraction in petunia flower-AN2 is a major determinant of pollinator attraction- Adaptation of plants to a new pollinator might be through single genes with large effects

Engineering Flavonoids with regulatory genes

Bovy et al., 2002


Bovy et al., 2002


Bovy et al., 2002


Bovy et al., 2002


Bovy et al., 2002


Bovy et al., 2002


- Using the regulatory genes LC/C1 from maize for overexpression in tomato

- A 60 fold increase in tomato flesh of the flavonol Kaempferol

- Flesh of tomato normally has very little flavonoids

- Both genes needed

Documents

Regulation of Flavonoid Biosynthesis (in seeds and other ... · Flavonoids in seeds Flavonols: ... Field tests for pollination Hoballah et al., 2007 Diurnal butterflies Diurnal hymenopteran